The formation and maintenance of appropriate and functional synaptic connections is a highly regulated process, with misregulation resulting in disordered cognition. Given the emerging information about gene expression regulation by non-coding RNAs, synaptic functions are likely to be regulated at least in part by non- coding RNAs, including microRNAs (miRNAs). Several miRNAs have been implicated in spinogenesis, dendritic arborization, and synaptogenesis. Thus, over- or under-expression of miRNAs in the brain could conceivably contribute to synaptic dysfunction resulting in neurological or neuropsychiatric disorders. Human chromosome 21 (HSA21) codes for 5 known miRNAs, and Trisomy 21 (TS21, i.e. Down syndrome) is the most common genetic form of intellectual disability. Thus, TS21 provides a unique model to study the effect of miRNA overexpression on the formation and functionality of synapses. The objective of this project is to elucidate the role of HSA21 miRNAs in synaptic dysfunction in human neurons generated from TS21 patients, which may be implicated in the cognitive disability in TS21 by testing the hypothesis that overexpression of HSA21 miRNAs leads to dysfunction in synaptic transmission that causes cognitive impairment in TS21 patients. Furthermore, there is preliminary evidence that suggests these miRNAs may affect synaptic integrity via a methyl CpG binding protein 2 (MeCP2) dependent pathway. Utilizing the innovative induced pluripotent stem (iPS) cell and induced neuronal (iN) cell technologies, it is possible to study the effects of HSA21 miRNAs on the synapses of human neurons. Following overexpression of HSA21 miRNAs in control iNs, synaptic function will be assessed by morphological and functional analyses, including electrophysiology and Calcium imaging. MeCP2 will be confirmed as a target of these miRNAs by dual luciferase assay, and its role in the miRNA-mediated modification of synapses will be tested via knockdown and rescue. Furthermore, patient-specific iNs will be used to elucidate whether the overexpression of HSA21 miRNAs causes synaptic defects in TS21. After verifying the expression levels of HSA21 miRNAs in TS21 iNs via qPCR and correlating them with the level of MeCP2 (determined by Western Blot), we will morphologically and functionally characterize the synapse for comparison with control iNs. We will then establish a cause-effect relationship between HSA21 miRNA overexpression and synaptic defects using Tough Decoys to antagonize the miRNAs and rescue the synaptic function of TS21-iN cells, as well as the expression level of MeCP2. The proposed research is innovative, because we will use interdisciplinary analytical methodologies and combine the newly developed iN cell and iPS cell technologies to examine the functions of HSA21 miRNAs in the nervous system, which will broaden our knowledge of their biological functions, as well as provide insight into mechanistic and molecular bases for the treatment of TS21.